2 Robertson et al. 973 temperature and precipitation changes is related to complex interactions between soil biota, water table depth, nutrient availability, root distribution, and soil and root respiration (Wurzburger et al. 2004). A better understanding of the ecological, economic, and social consequences of plant migration may be gained through studies directed below ground, as the functioning of terrestrial ecosystems and their ability to adapt to environmental changes appears to depend on soil communities (Dahlberg 2001; Hart et al. 2001; Prosser 2002). Ectomycorrhizas (ECMs) are essential to ecosystem functions including biogeochemical cycling, maintaining soil structure and forest food webs, and buffering plants against environmental stress (Brundrett 1991; Colpaert and van Tichelen 1994). The structure (species composition) and diversity (species richness and abundance) of ECM fungal communities vary across landscapes according to host specificity and edaphic factors (Molina et al. 1992; Allen et al. 2003). For example, Gehring et al. (1998) reported a change in pinyon pine (Pinus edulis Engelm.) ECM community composition across a soil moisture gradient, but no significant difference in species richness. The ECM community composition of white spruce (Picea glauca (Moench) Voss), Norway spruce (Picea abies (L.) Karst.), and European beech (Fagus sylvatica L.) also varied across nitrogen (N) deposition gradients, accompanied by a decline in ECM species richness with increasing soil N availability (Taylor et al. 2000; Lilleskov et al. 2002). Very few studies have attempted to relate ECM distribution to environmental gradients independent of host plant (O Dell et al. 1999). Black spruce (Picea mariana (Mill.) BSP), a dominant species of the Canadian boreal forest, exhibits broad tolerance to soil moisture levels. It typically grows in Sphagnumdominated wetlands characterized by cold, poorly aerated peat soils with low nutrient availability, either in pure stands or mixed with tamarack (Larix laricina (Du Roi) K. Koch) (MacKenzie and Moran 2004). Along peatland gradients in Wisconsin, larch was found to dominate the wetter, more minerotrophic (fed by mineral-rich ground water) sites, whereas black spruce dominated drier, more ombrotrophic (fed by very nutrient-poor precipitation) sites (Montague and Givnish 1995). Black spruce is well adapted to watersaturated soil conditions, establishing on hummocks of woody debris and producing shallow roots that spread laterally (Lieffers and Rothwell 1987; Roy et al. 1999). Despite low dissolved oxygen and reducing conditions in wetland soils that may inhibit ECM formation by limiting fungal metabolism (Walker 1987), many woody plant species (including black spruce) in northern wetlands have been found to be ectomycorrhizal (Thormann et al. 1999; Wurzburger et al. 2004). In adjacent upland forests, black spruce associates with lodgepole pine (Pinus contorta Dougl. ex Loud. var. latifolia), as well as with other tree, shrub, and herb species that generally indicate low to medium productivity plant communities developed over nutrient-poor, water-deficient soils (Krestov et al. 2000). Drier upland soils often exhibit a root-restricting clay horizon near the surface that perches the water table during wet periods of the year, leading to seasonal cycles of moisture surplus and deficit (Krestov et al. 2000). Upland trees often appear more robust than their wetland counterparts; however, improved edaphic conditions do not seem to result in ecotypic variation in black spruce morphological features or water relations (Parker et al. 1983; Zine El Abidine et al. 1994). Results from greenhouse and field experiments with Norway spruce (Picea abies (L.) Karst.) suggest that survival in seasonally dry soils may be due to an increased branching density of fine roots in response to water stress, allowing for uptake of water from dry soil via ECM (Feil et al. 1988). The presence of ECM on naturally regenerating and outplanted black spruce has been confirmed in upland habitats (Malloch and Malloch 1981; McAfee and Fortin 1989), but the ECM communities associated with this host species have not been previously described. The objective of this study was to describe the ECM fungal communities associating with naturally regenerating black spruce seedlings from forest sites demarcating wet and dry habitats in central British Columbia. Sites included black spruce dominated wetlands, black spruce tamarack wetlands, and black spruce lodgepole pine uplands, which represent typical black spruce habitats that comprise a substantial component of the Canadian boreal forest (Burton et al. 2003). A combination of morphological (light microscopy) and molecular (polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP)) techniques were used to characterize the structure and diversity of ECM fungal communities and to assess potential differences between wetland and upland habitats that varied with respect to dominant vegetation and soil properties. PCR-RFLP results were also used to evaluate intraspecific variation (genotypic variation within characterized ECM morphotypes) for the ECM fungal community. We expected that some ECM fungi would be associated with black spruce across all three habitats, but that variable moisture and soil conditions and associated vegetation changes would contribute to community differences at both the morphotype and genotype levels. We also predicted lower diversity in the wetland habitats due to the stress imposed by water saturation of soils. Insights into ECM community variation across environmental gradients should provide a better understanding of how climate change will impact boreal forest landscapes and whether ECM systems might facilitate or mediate the movement of plant associations in response to change. Materials and methods Study area The study area was located in the Sub-Boreal Spruce (SBS) biogeoclimatic zone in the central interior of British Columbia. The SBS ranges from N latitude and an elevation of m (Meidinger et al. 1991; DeLong and Fahlman 1996). Climate is characterized by severe and snowy winters, warm, moist, and short summers, and moderate annual precipitation (Meidinger et al. 1991). Site descriptions Three replicate sites were identified within each of three forest habitat types: black spruce dominated wetlands (W), mixed black spruce tamarack wetlands (T), and mixed black spruce lodgepole pine upland forests (U). The W sites, classified as black spruce water sedge peat moss bogs / poor fens (Wb05, MacKenzie and Moran 2004), were located approximately 60 km northwest of Prince George,

3 974 Can. J. For. Res. Vol. 36, 2006 British Columbia, in the moist, cool subzone variant (SBSmk1) of the SBS. These bogs were dominated by mature black spruce stands with an understory of scrub birch (Betula glandulosa var. glandulosa Michx.), willow (Salix spp.), Labrador tea (Ledum groenlandicum Oeder), bog cranberry (Vaccinium oxycoccos L. MacM.), and bog rosemary (Andromeda polifolia L.). The T sites, classified as tamarack water sedge fen moss bogs / poor fens (Wb06, MacKenzie and Moran 2004), were located approximately 40 km west of Prince George, in the dry, warm (SBSdw3) subzone variant of the SBS. Tamarack and black spruce were the dominant tree species, and the shrub understory consisted mainly of scrub birch, willow, and Labrador tea. In both wetland habitat types, site microtopography consisted of raised mounds (hummocks of partially decomposed moss that covered woody debris) amongst depressions (hollows) that were often below the level of the water table. Sphagnum moss covered most of the open areas, with other mosses and lichens (Cladina and Peltigera species) on drier hummocks and woody debris. Soils were classified as Typic Humisols, a subgroup of the Organic soils (Soil Classification Working Group 1998). The U sites were located adjacent to the black spruce dominated wetlands (W sites). These sites were dominated by mature black spruce and lodgepole pine, with a small component of hybrid white spruce (Picea glauca Picea engelmannii Parry ex Engelm.), subalpine fir (Abies lasiocarpa (Hook.) Nutt.), or sitka alder (Alnus crispa var. sinuata (Reg.) Rydb.). The understory contained a diversity of shrubs and herbs, including prickly rose (Rosa acicularis Lindl.), pink spirea (Spiraea douglasii subsp. menziesii Hook.), black twinberry (Lonicera involucrata (Richards.) Banks ex Spreng.), highbush cranberry (Viburnum edule (Michx.) Raf.), twinflower (Linnaea borealis L.), dwarf blueberry (Vaccinium caespitosum Michx.), trailing raspberry (Rubus pubescens Raf.), kinnikinnick (Arctostaphylos uva-ursi L. Spreng.), round-leaved rein-orchid (Platanthera orbiculata), rattlesnake plantain (Goodyera oblongifolia Raf.), one-sided wintergreen (Orthilia secunda (L.) House), pink wintergreen (Pyrola asarifolia Michx.), and bunchberry (Cornus canadensis L.); mosses and lichens formed the forest floor. Upland soils were classified as Orthic Gray Luvisols, which characteristically develop in well to imperfectly drained sites with sandy loam to clay soils under boreal or mixed forests (Soil Classification Working Group 1998). Seedling, soil, and sporocarp sampling Sampling plots (50 m 50m)were established at least 10 m inside site boundaries at each of the nine sites. All regenerating (no obvious signs of vegetative layering) black spruce seedlings (15 30 cm in height) were located within each plot. Five seedlings were randomly selected from each plot (total of 45 seedlings) and harvested (along with the surrounding soil) between 26 July and 2 August 2001, when optimal fine root growth and ECM development was expected to have occurred. Seedlings were placed in pots, bagged, and stored at 5 C until ECM characterization was performed. Organic soil samples were collected from several locations within each wetland plot, combined for each plot, and stored in plastic bags. Two or three soil pits were excavated within upland forest plots, and samples from the forest floor and mineral horizons were collected, combined for each plot, and bagged. Soil samples were dried and stored at 22 C for total carbon (C), total N, extractable phosphorus (P), and ph analyses (Kalra and Maynard 1991). Fungal sporocarps were collected from all study sites at the time of seedling harvest. After descriptions and fungal identities were recorded, samples were taken from the sporeproducing tissues and stored in microcentrifuge tubes at 20 C for DNA analysis and subsequent comparisons to ECM root tip data. ECM characterization Root systems were gently washed free of soil, mosses, and debris in tap water, and cleaned roots were floated over a grid of 2 cm 2 cells in a tray of water. Root fragments 2 cm long were randomly selected from cells until approximately 200 root tips had been sampled. One unbranched root tip was considered to be one ECM. Only roots that appeared turgid with intact meristems were examined. ECM tips were initially described using a dissecting microscope and classified according to color, texture, lustre, dimensions, tip shape, branching pattern, and presence or absence of rhizomorphs (mycelial strands) following standard techniques of Agerer ( ), Ingleby et al. (1990), and Goodman et al. (1996). Root squash mounts were subsequently examined using bright field microscopy, and descriptions of mantle features, emanating hyphae, rhizomorphs, and other characteristics were used to further categorize the different ECM morphotypes. Root tips that appeared uncolonized or lightly colonized (due to the lack of a well-developed mantle) were classified as nonmycorrhizal. Identification to family or genus was made based on similarities in features to published descriptions (Agerer ; Ingleby et al. 1990; Goodman et al. 1996); when identification was not possible, a descriptive name was assigned. DNA extraction, amplification, and restriction endonuclease digestion From each seedling, a proportionally representative sample of 20 root tips (10% of each morphotype) was stored at 20 C for molecular analysis. DNA was extracted from tips using a modified CTAB (2 hexadecyl trimethyl ammonium bromide) extraction protocol (Mah et al. 2001). PCR amplification of the internal transcribed spacer (ITS) region of fungal rdna was conducted using Platinum Taq DNA polymerase (Invitrogen, Burlington, Ontario) along with the universal oligonucleotide primer ITS1 (White et al. 1990) and the fungal-specific primer NL6Bmun (Egger 1995). Reaction tubes were placed in a PTC-100 TM programmable thermal controller (MJ Research, Inc., Waltham, Massachusetts), programmed to the following settings: denaturation at 94 C for 30 s and 93 C for 35 s, annealing at C for 53 s, and extension at 72 C beginning at 30 s and increasing by 5 s per cycle for 35 cycles. PCR products were digested using the restriction endonucleases AluI, HinfI, and RsaI (Invitrogen). Resulting fragments were run with 1 kb DNA ladder (Invitrogen) for approximately 3honhigh-resolution gels (1.0% agarose plus 1.5% low melting point agarose in 10% Tris borate EDTA (TBE) buffer) containing ethidium bromide for visualization at ~115 mv. Digital images were

4 Robertson et al. 975 saved using the Gel Print 2000I photographic system (BioPhotonic Corp., Dexter, Michigan). Data analysis Soil nutrient content (C, N, and P) and ph were compared between habitats (and between different soil horizons in the U sites) using one-way analysis of variance (ANOVA) (Systat version 8.0, SPSS Inc., Chicago, Illinois). Mean comparisons were tested using Fisher s least significant difference (LSD) test (α = 0.05). ECM frequency of occurrence (number of seedlings on which each morphotype was found) and abundance (proportion of each morphotype on a seedling) was calculated for each seedling, site, and habitat type. The relative abundance of each morphotype was plotted against species rank order to visualize and compare ECM community structure between habitats (Magurran 1988; Taylor et al. 2000). Mean abundance values for individual morphotypes (calculated per seedling and averaged within each habitat) were compared between habitats using one-way ANOVA and Fisher s LSD test (α = 0.05). ECM diversity was measured using the Margalef, Shannon, Shannon evenness, and Simpson indices (Magurran 1988). Diversity indices were calculated from morphological data for each seedling and averaged within habitats. To determine differences between habitats, indices were compared using one-way ANOVA and Fisher s LSD test (α = 0.05). RFLP gel images were analyzed using RFLPscan version 3.12 (Scanalytics 1994). Individual DNA fragments were marked and their sizes (base pairs) calibrated against the 1 kb standards (1018, 514, 356, 344, 298, 220, 201, 154, 134, and 75 bp fragments) using the Desmile calibration method with log piecewise linear curve fitting. Fragments smaller than 80 bp were excluded from analysis to reduce the possibility of primer dimer products being included. Fragment patterns for individual samples were imported into both individual morphotype and seedling databases in RFLPscan Database version 3.12 (Scanalytics 1994). Fragment patterns obtained from digestion of field-collected sporocarp samples were imported into a separate database for comparison. Within each RFLPscan database, pairwise comparisons of all band patterns were compiled for each of the three enzymes. Band patterns were matched at a 5% threshold, a level determined to represent the best balance between type I (incorrectly calling the same fragment different) versus type II (incorrectly calling different fragments the same) errors (Baldwin 1999). Values ranged from zero to one within each enzyme and from zero to three between two samples, with three indicating an identical ECM. The fragment pattern data were imported into a spreadsheet, where samples were first sorted into morphotypes, then into genotypes. A genotype was defined as a set of fragment patterns that, when compared to other sets, is within a 5% threshold of similarity for all three enzyme digests. Partially digested or potentially contaminated samples were removed from the total sample and average fragment sizes were calculated for each genotype. Cluster analysis of the similarity matrix using the neighbour-joining method was performed using PHYLIP (Phylogeny Inference Package) version 3.573c (J. Felsenstein, University of Washington), and resulting phylograms were viewed in TreeView version Win 3.2 (Roderick D.M. Page, Institute of Biomedical and Life Sciences, University of Glasgow). Individual databases were merged into one large database, which was compared to reference ECM root tip (K. Egger, unpublished data) and sporocarp databases to confirm the classification and taxonomic naming of ECM morphotypes and to attempt to identify unknowns based on clustering with known types. From all successfully digested root tips, pairwise matching of restriction fragment patterns was also conducted using Dice s index (Dice 1945), which was then converted to a distance matrix (1 Dice s index) to calculate the phi (Φ) index (Mah et al. 2001). Phi values were used to assess molecular diversity within each morphotype (intraspecific diversity) and between habitats (data pooled for sites) using one-way ANOVA and Fisher s LSD (α = 0.05). Results Seedling and soil characteristics No differences in seedling characteristics (i.e., height and age) were observed between habitats. Although only seedlings considered to be naturally regenerating (true seedlings) were selected for harvest, almost half (44.4%) of the harvested seedlings were found to originate from branches of trees that had developed underground adventitious roots and formed new seedlings (layered seedlings). These were evenly distributed across habitats. Soil nutrient content (C, N, and P) varied significantly between the wetland (both T and W) and upland habitats. Total C and total N content were greater (p = and 0.001, respectively) in the wetland soils, whereas the C:N ratio and available P were greater (p = and 0.025, respectively) in the upland forest floor soils. All soil samples were acidic (ranging from ph 3.8 to 6.9), and ph did not vary significantly between wetland and upland soils. ECM morphotype occurrence, frequency, and abundance Overall, 85% of all root tips examined were ectomycorrhizal. The majority (~30) of the 33 ECM morphotypes described from 8858 root tips were likely basidiomycetes. Twenty-seven morphotypes were assigned to families including the Thelephoraceae, Cortinariaceae, and Russulaceae, or to genera such as Cenococcum, Mycelium radicis atrovirens (MRA), Amphinema, Lactarius, Tomentella, Piloderma, and Hebeloma. Six morphotypes could not be assigned with confidence to any taxonomic group. Detailed morphological descriptions are in Robertson (2003). Nonmycorrhizal tips (1337) were found on 66.7% of all seedlings (including seedlings from each habitat and from all sites) with significantly greater numbers in the W habitat (p = 0.000). Between 2 and 8 (mean of 4.8) ECM morphotypes were identified per seedling. The mean number of morphotypes varied significantly (p = 0.005) between habitats, with more occurring in the two mixed-species habitats (mean of 5.2 for both the T and U habitats) than in the black spruce dominated habitat (mean of 4.0 morphotypes). The number of morphotypes found on true seedlings (5.16 ± 0.24, n = 25) was significantly greater (p = 0.037) than the number that occurred on layered seedlings (4.35 ± 0.29, n = 20).

5 976 Can. J. For. Res. Vol. 36, 2006 Fig. 1. Comparison of rank-abundance of ECMs of black spruce in three habitats (black spruce tamarack wetlands (T), black spruce dominated wetlands (W), and black spruce pine upland forests (U)), using log abundance plotted against ranked morphotype abundance for each habitat. log (number of root tips) T Sites W Sites U Sites Morphotype abundance rank ECM communities in each habitat consisted of a few dominant fungal species and many rare species (Fig. 1). ECM morphotype log rank abundance curves appear fairly even for wetland habitats; for the upland habitat, the curve tends toward a broken stick distribution (i.e., more distinct drops with decreasing ECM abundance), which can be interpreted as representing the lack of a relationship between different ECM types, with each species obtaining a random fraction of the total resource (Magurran 1988). Wetland habitats were dominated by Cenococcum, Lactarius 1, Amphinema (T sites), and Thelephoraceae-like 4 (W sites) morphotypes, whereas Cenococcum, Cortinariaceae 2, Russulaceae 2, and Amphinema morphotypes dominated in the upland habitat. Table 1 shows the comparison of the frequency of occurrence and relative abundance of all ECM morphotypes between habitats. Abundance varied significantly between habitats for 6 of the 14 ECM morphotypes that occurred on four or more seedlings. Russulaceae 2 and Piloderma were significantly more abundant (p = and p = 0.001, respectively) on upland seedlings than on wetland (T or W site) seedlings. Tomentella and Tomentella-like 1 were more abundant (p = and p = 0.037, respectively) in the black spruce tamarack wetlands than in either of the other habitats. Lactarius 1 ECM abundance was also significantly greater (p = 0.047) in the black spruce tamarack wetlands than in the upland habitat (where it was not found) and greater (but not significant) than in the spruce-dominated wetlands. Thelephoraceae-like 4 was the only morphotype that was significantly greater in the spruce-dominated wetlands than in either of the mixed-species habitats (p = 0.025). Although differences in ECM frequency occurred between habitats for some commonly occurring morphotypes such as Cenococcum, Cortinariaceae 1, Cortinariaceae 2, Amphinema, and MRA 1, mean abundance values did not vary significantly. Molecular fragment pattern analysis and diversity within morphotypes From the original ECM root tips sampled for PCR-RFLP analysis (888), 51% (454) yielded restriction fragment patterns suitable for molecular analysis. The success rate from colonized tips (755) was 60% and varied with morphotype. Types such as Amphinema and Piloderma had higher success rates (>75%) than Cortinariaceae and Thelephoraceaelike types (37% 48%). With respect to nonmycorrhizal tips (133 samples), 24% (32) amplified and produced fungal DNA fragment patterns. Of these, 26 matched patterns of Cenococcum (genotypes 1 and 2), MRA 1 (genotypes 2 and 3), Amphinema (genotype 3), Russulaceae 1 (genotype 1), Russulaceae 2 (genotypes 1 and 6), and Thelephoraceae-like 2 (genotype 2). Cluster analysis of fragment patterns generally confirmed the morphological classification of ECM morphotypes (tree not shown), but fungal identification (as related to taxonomic placement) was generally not improved. Comparisons with ECM root tip reference databases also helped to establish taxonomic groupings. For example, one group classified as nonmycorrhizal tips clustered with a reference sample of E-strain, allowing tentative identification of these ECMs as a type not morphologically described in this study. When fragment patterns generated from ECM root tips were compared to those for field-collected sporocarps, few matches were found. Two exceptions were Lactarius deliciosus (Fries) S.F. Gray, which grouped within the Lactarius 1 genotype 1 clade, and Lactarius torminosus (Schaeff. ex Fr.) Gray, which grouped within the Lactarius 3 genotype 1 clade. Averaged restriction fragment lengths for all ECM genotypes are presented in Table 2. Most morphotypes consisted of several genotypes. Morphotypes with the most genotypes included Amphinema (six), Cortinariaceae 2 (six), and Russulaceae 2 (seven); Cenococcum (two); Thelephoraceaelike 1 and Piloderma only exhibited one genotype. In a few cases, delimitation of genotypes was based on fragment size variation in only one of the three enzyme digests. For example, Amphinema genotypes 1, 2, and 3 varied only by the AluI pattern; genotype 5 differed from genotype 1 only by the RsaI enzyme. The single genotype of Lactarius 3 varied from Lactarius 1 genotype 1 only by the HinfI pattern, as both profiles showed fragments that were not digested by the

7 978 Can. J. For. Res. Vol. 36, 2006 one other genotype at T sites and one genotype at both the W and U sites. When larger taxonomic groups were considered, other habitat distribution patterns were observed. For the Cortinariaceae group, the number of genotypes was fairly even across all habitats. In contrast, more Lactarius genotypes were in the T habitat (four) than in either the W (two) or U (one) habitats. Half of Russulaceae genotypes occurred in the U habitat (eight), followed by the T (five) and W (two) wetland habitats. The majority of genotypes (88%) from the Thelephoraceae, Tomentella, and brown types were found in the wetland habitats; Tomentella genotypes appeared restricted to the T sites. In some instances, ECM genotype distribution patterns mirrored ECM morphotype occurrence (where ECM are present at some sites and not others). Habitat effects on ECM morphotype and genotype diversity All diversity indices (Margalef, Shannon, Shannon evenness, Simpson, and Φ) indicated that ECM community diversity was greatest in the black spruce pine upland (U) forests and least in the spruce-dominated wetland (W), with intermediate diversity in the spruce tamarack wetland (T) habitat (Table 3). Species richness (Margalef index) varied significantly (p = 0.038) between the black spruce dominated wetlands and both mixed habitats, which had similar and higher richness values. Shannon index values were also significantly greater (p = 0.028) in the black spruce pine upland habitat than in the black spruce dominated wetland habitat, whereas tamarack black spruce wetlands had intermediate values. Shannon evenness and Simpson indices did not vary significantly, but supported similar trends. Molecular diversity did not differ significantly between habitats, although Φ index values were higher in the T and U habitats than in the spruce-dominated wetlands. Discussion ECM morphotype occurrence and abundance Although the ECM status of black spruce has been confirmed by others (Malloch and Malloch 1981; Thormann et al. 1999), this is the first study to report on the composition and diversity of black spruce ECM communities across a range of habitats representing a moisture gradient. The 33 ECM morphotypes characterized for black spruce is within an expected range (20 35 fungal species) for small monoculture forests in relatively homogeneous environments (Bruns 1995). In central British Columbia, Mah et al. (2001) described 24 ECM morphotypes on naturally regenerating and planted hybrid spruce seedlings from clearcut or burned and mature SBS forests, while Kranabetter et al. (1999) reported as many as 74 morphotypes (an average of 52) on 2-year-old outplanted white spruce, subalpine fir, and lodgepole pine seedlings. Flynn et al. (1998) described 13 morphotypes on naturally regenerating Sitka spruce (Picea sitchensis (Bong.) Carr.) seedlings from Scottish plantation forests. Reported variation in ECM diversity may be attributed to differences in host receptivity to ECM fungi, inoculum potential at sites, soil and environmental factors, sampling intensity, or differences in techniques used by the investigators. The relatively high number of morphotypes in the present study reflects variation in the ECM community composition across the three habitats (including host species composition) and suggests the importance of sampling across the ecological range of any host to capture associated differences. Many of the ECM communities described in this study consist of fungal taxa that have extensive distributions with little host specificity and that often associate with N-limited, acidic forest soils (Molina et al. 1992; Goodman and Trofymow 1998). Although most ECM morphotypes were basidiomycetes, ascomycete fungi (predominantly Cenococcum and MRA 1) accounted for almost 19% of the overall community. Malloch and Malloch (1981) found that Cenococcum was frequently associated with black spruce roots collected from upland forests in northeastern Ontario, and Thormann et al. (1999) identified Cenococcum and MRA on black spruce roots from bogs and fens in northern Alberta. Cenococcum geophilum Fr. is considered a cosmopolitan fungus with a wide host range and broad environmental tolerances (Goodman and Trofymow 1998). Less is known about fungi in the MRA species complex, but some MRA types have been described as exhibiting both mycorrhizal and pathogenic characteristics (Summerbell 1989; De la Bastide and Kendrick 1990). ECM abundance did not vary significantly across the moisture gradient for either Cenococcum or MRA, but their presence in both wetland types suggests a level of tolerance by these fungi for wetter habitat associations. ECM abundance for the Cortinariaceae and Amphinema morphotypes was similar between wet and dry sites; other results support this lack of habitat specificity exhibited by Amphinema, but not for Cortinariaceae types. Lilleskov et al. (2002) found that along a N deposition gradient, Amphinema (A. byssoides Pers.: Fr.) white spruce mycorrhizas occurred on sites having low to intermediate soil N in contrast to Cortinarius species that were confined to low-n sites. Both upland and wetland habitats in this study were likely N limited (the highest N content found on any site was 2.6%) and could, therefore, support both Amphinema and Cortinariaceae ECM fungi. Several Russulaceae, Thelephoraceae, and Piloderma morphotypes exhibited habitat specificity. Fungi belonging to Russulaceae and Lactarius types were identified across all habitats; however, Russulaceae 2 was significantly more abundant at the drier upland sites than was Lactarius 1 (Lactarius deliciosus), which was most abundant at the spruce tamarack wetlands and not found in the upland habitats (even though it has been described as being environmentally tolerant and lacking substrate specificity) (Goodman and Trofymow 1998). Remaining Russulaceae and Lactarius morphotypes occurred in relatively low abundance, usually specific to single upland or black spruce tamarack habitats. In contrast, Lilleskov et al. (2002) found that Lactarius white spruce ECM were less abundant at low-n than at high-n sites. Although our sites appeared to be N limited, total N was greater in the wetland habitats, where it was likely retained in organic forms. We do not know if Lactarius or Russulaceae fungi are able to use organic forms of N or to adapt to N-limiting conditions. Thelephoraceae morphotypes (including Tomentella and several unidentified brown types) were identified most often in

10 Robertson et al. 981 Table 2. (concluded). Habitat Approximate fragment sizes (bp) ECM genotype T W U No. Size (bp) AluI HinfI RsaI Genotype Creamy rhizomorphic clamped Genotype Genotype Orange 1 Genotype Brown 1 Genotype Brown 3 Genotype E-strain Genotype Note: DNA fragments from amplified fungal rdna using the ITS1 and NL6Bmun primers, and digestion with the restriction endonucleases AluI, HinfI, and RsaI. No. refers to the number of tips amplified. Size refers to the length of undigested rdna. both wetland habitats; three frequently occurring morphotypes (Tomentella, Tomentella-like 1, and Thelephoraceae-like 4) were significantly more abundant at wetland than at dry upland sites. Tomentella closely matched an uncommon variant of the Tomentella-like with cystidia morphotype described by Danielson and Pruden (1989) on urban blue spruce (Picea pungens Engelm.) and white spruce. The color, mantle features, and cystidia of Tomentella-like 1 were similar to those of dark-mantled types of Tomentella (Agerer ; Lilleskov et al. 2002) and those described from black spruce peatlands in northern Alberta (Thormann et al. 1999). Thelephoraceae conifer associations are well known and important components of ECM communities (Kõljalg et al. 2000). Many form inconspicuous resupinate fruit bodies on dead plants, wood, and soil debris (Kõljalg 1996), and some have been functionally classified as decomposers (Read and Perez-Moreno 2003). The ability of some fungi in the thelephoroid group to revert to a free-living saprotrophic state may give these fungi a competitive advantage over nondegrading ECM fungi in nutrient-poor habitats (Hibbett et al. 2000). Although much remains to be explored with respect to this group of fungi, Thelephoraceae had a propensity towards forming numerous symbioses with black spruce in the very wet sites. Piloderma (Piloderma fallax (=croceum) Erikss. & Hjortst.) was identified only from the upland sites, almost exclusively on seedlings rooted in coarse woody debris. Others have noted the strong relationship between the occurrence of Piloderma and the percent cover of advanced-decay coarse woody debris (Goodman and Trofymow 1998), as well as its association with acidic humus and forest floor mineral soil boundary layer (Arocena et al. 1999). Piloderma species may be able to adapt to relatively acidic, N-poor soil conditions and specialize in efficient N uptake (Lilleskov et al. 2002) or increase nutrient availability through enzymatic degradation of plant material (Griffiths and Caldwell 1992). Nevertheless, despite the abundant coarse woody debris on low-n, acidic wetland sites, where seedlings often grew, Piloderma was never found. Factors such as soil saturation, low oxygen, and cold substrates may inhibit Piloderma growth on these sites. Molecular variation within ECM morphotypes Sixty-five genotypes were delimited from the 29 ECM morphotypes successfully amplified and digested. Others using similar methods have reported comparable orders of magnitude: 22 genotypes were described for the 8 most common ECM morphotypes on hybrid spruce (Mah et al. 2001), 26 genotypes for 11 morphotypes of Douglas-fir (Sakakibara et al. 2002), and 23 genotypes within 18 morphotypes on Norway spruce (Mehmann et al. 1995). In the present study, some restriction fragment patterns (comparing two or three enzymes) of Amphinema, Cenococcum, Lactarius 1, MRA 1, and Piloderma were similar to those reported by Mah et al. (2001) and Sakakibara et al. (2002). The number of fragment patterns (genotypes) within morphotypes varied from low for Piloderma (one genotype) and Cenococcum (two) to high for Amphinema (six), Cortinariaceae 2 (six) and Russulaceae 2 (seven). Mah et al. (2001) found similar intraspecific variation (i.e., genotype variation within morphotypes) for Cenococcum (one) and

11 982 Can. J. For. Res. Vol. 36, 2006 Table 3. ANOVA for mean diversity values (standard error in parentheses) for ECM morphotypes (Margalef, Shannon Evenness and Simpson indices) and genotypes (Phi index) showing habitat effects for three black spruce communities. Treatment effect Diversity index F P Tamarack spruce wetland Black spruce wetland Pine spruce upland Margalef (0.071)a (0.059)b (0.045)a Shannon (0.085)ab (0.110)b (0.070)a Shannon evenness (0.038) (0.055) (0.040) Simpson (0.217) (0.268) (0.243) Phi (0.035) (0.027) (0.031) Note: Mean comparisons were tested using Fisher s least significant difference test. Means followed by the same letter are not significantly different (α = 0.05). P values 0.05 are presented in bold. Amphinema (six). In contrast, Sakakibara et al. (2002) found low variation within most morphotypes and many that expressed one dominant set of fragment patterns. Consistent with other studies (Gehring et al. 1998; Horton 2002), we report morphotypes having one or two dominant restriction fragment patterns as well as those with more even distributions of genotypes. Differences may result from a lack of variation in the ITS region of some ECMs; however, minor variations in analysis techniques and decisions made during morphotyping (e.g., classification of morphotypes as the same or different), larger sample sizes, or assignment of restriction fragment patterns as genotypes may also alter outcomes. Genotype variation appeared higher in the morphotypes that were less well defined (such as Cortinariaceae and Russulaceae), some of which grouped in several polyphyletic clusters on the neighbour-joining tree and may represent different species. Intraspecific diversity for ECMs as measured by the Φ diversity index was lowest for Piloderma and Cenococcum morphotypes and highest for MRA 1, Thelephoraceae-like 4, and Tomentella-like 1. Although the latter three ECMs did not have the greatest number of genotypes, high Φ values reflect greater pairwise distances between restriction fragment patterns. Russulaceae 2 and Lactarius 1 also had high diversity values, whereas Amphinema and Cortinariaceae 1 and 2 had intermediate values. The index suggests that, although Amphinema and Cortinariaceae 2 were in the group having the highest number of genotypes, their intraspecific variation was less than that for several other morphotypes. Calculating Φ diversity values is less subjective when assessing intraspecific variation than assigning genotypes to restriction fragment patterns. Habitat effects on ECM community diversity ECM community diversity was highest in the black spruce lodgepole pine upland forests and lowest in the black spruce dominated wetlands; differences were significant when measuring species richness (Shannon and Margalef indices). The Shannon evenness and Simpson indices (reflecting morphotype abundance), as well as the Φ index (a measure of molecular diversity), supported this trend but were not significant. Higher species richness in the drier upland habitat than in both wetland habitats may reflect improved edaphic conditions, particularly soils that are not water saturated for most of the growing season. In general, the lack of aeration is expected to slow or inhibit ECM formation in wetland soils (Walker 1987), although in vitro studies of inoculated jack pine (Pinus sylvestris Lamb.) seedlings showed that different ECM fungal species varied in their susceptibility to periodic flooding (Stenström 1991). It is unknown whether this contributed to the greater proportion of nonmycorrhizal roots observed in black spruce dominated wetlands than in both upland forests and black spruce tamarack wetlands in this study. The propensity of black spruce seedlings to establish on Sphagnum hummocks, as well as observations that the water table receded during the period of ECM establishment, indicate that oxygen deficiency was likely not a major factor in determining the composition of ECM communities on wetland black spruce. Soil nutrient availability may also be altered by water saturation. Although total N was greater in wetland soils, much of this was likely in less accessible organic forms because of low rates of decomposition in these cold, wet habitats (Thormann et al. 1999). Fungal symbionts that are physiologically able to capture nutrients from organic sources (Read and Perez-Moreno 2003) may explain the greater diversity in Thelephoraceae types in the wetland habitats. Phosphorus availability, also low compared to levels in upland soils, may be less important than N availability in ECM systems (Smith and Read 1997), but it is expected that ECM fungi in P-limited habitats would also be able to maximize assimilation of P for their plant partners. Roots of large, dominant companion plants (tamarack and lodgepole pine) can act as refuges for fungal inoculum for growing black spruce roots and most likely contributed greatly to higher diversity in both mixed-forest habitats. ECM establishing on the root systems of alternate hosts such as pine or larch species can increase the inoculum available to neighbouring plants (Walker 1987; Massicotte et al. 1999). Jonsson et al. (1999) have shown the importance of large root systems as a source of inoculum, citing similarities in species composition of ECM fungi colonizing naturally regenerating pine seedlings and mature trees in Swedish boreal forests. Overall, ECM morphotype abundance showed the expected dominance by a few ECM types, with other morphotypes occurring in progressively fewer numbers (Horton and Bruns 2001; Taylor 2002). Each habitat type, irrespective of moisture or associated host species, had approximately 20 ECM morphotypes, of which about half were dominant fungi; rare types were generally confined to one or two habitats. The ability of ECM fungi to dominate a host s root system has led some to suggest that species richness may be less important than the dominant species present because it includes a level of functional redundancy that is expected to exist within the soil microbial community (Hart et al. 2001). Nev-

12 Robertson et al. 983 ertheless, rare species are a widely reported component of ECM fungal communities and future studies should continue to examine the contributions of rare species to ecosystem processes, particularly in light of changing temperature and precipitation patterns. Among genotypes of the more generalist ECM fungi, the molecular analysis provided some resolution of habitat specificity. Cenococcum, MRA 1, and Cortinariaceae 1 morphotypes and genotypes occurred across both wet and dry habitats, whereas genotypes of Amphinema, Cortinariaceae 2, and Russulaceae 2 showed more uneven patterns of distribution. Habitat- and site-specific associations have also been reported for Amphinema and MRA hybrid spruce ECMs, lodgepole pine ECMs, and tomentelloid types based on RFLP profiles and ITS sequences (Byrd et al. 2000; Kõljalg et al. 2000; Mah et al. 2001). The tendency of the rank-abundance model (Fig. 1) towards a broken stick distribution for ECMs in the upland habitat may be interpreted to reflect greater soil heterogeneity, which is considered a major factor in maintaining high diversity in ECM communities (Bruns 1995; Taylor 2002; Allen et al. 2003). Examination of the spatial distribution of ECM genotypes across environmental gradients, when sample sizes are large, can contribute to increased incidental distribution information that may be lost in studies with smaller sample sizes. Kernaghan (2001) found that their small molecular sample was suitable for confirming ECM identities (through fragment pattern comparisons), but too small to detect genotype variation between sites. The ITS region is expected to show intraspecific variation across large geographic scales due to its rapid rate of evolution and the reproductive isolation of populations in different habitats (Horton 2002). However, based on a small sporocarp sample, intraspecific variation in the ITS region has been reported to be negligible at local scales (Kårén et al. 1997). If some intraspecific variation is tracking adaptation of populations to changing environmental conditions, it seems probable that variation in the ITS region could occur at various geographic scales in response to environmental heterogeneity and selective pressures (Cairney 1999). The ability to measure ECM richness and abundance is related to sampling effort (number of tips), probability of detection, and the relative abundance of each species (percentage of root tips colonized) (Taylor 2002). Compared to the sample size of many studies, our sample size should have detected most ECM types in the habitats, at least for black spruce in an early stage of development. Horton and Bruns (2001) emphasized the clustered distribution of many ECM fungi and reported that most actually occur in fewer than 10% of all samples. Clustered distributions of ECM morphotypes may be partly due to the patchy distribution of habitat features and may explain why morphotype richness is much lower at the individual seedling level than at the site or habitat level. In the present study, morphological and molecular analyses suggest a mosaic of black spruce ECM associations consisting of those occurring in both wetland and dry upland forest types, as well as those limited to one or two of the habitats. This mix of shared and habitat-limited associations most likely results from a combination of factors that include moisture differences, variable site hosts, and other soil attributes; the ability of black spruce to shift between fungal associates over diverse landscape and conditions suggests that this species will be able to accommodate a range of climatic changes. Black spruce currently occupies vast tracts of the boreal and sub-boreal forest, where it is subject to varying moisture and temperature gradients. This study supports the hypothesis that alternate hosts, and their ECM associations, increase and (or) modify those ECM associations of black spruce, although it remains unclear how these might be impacted by climate change. Most certainly it will depend partly on the ability of these alternate hosts to adjust to abiotic and biotic shifts. Nevertheless, based upon this study, black spruce, with ECM fungal associates adapted to both wet and dry habitats, seems to be well positioned to tolerate climate changes. Patterns of adaptation may be determined more by the rate at which climate factors change as well as how the extended biotic community, as a whole, is also able to respond to these changes. A greater understanding of the functional aspects of ECM diversity in forest processes will facilitate our ability to assess the impacts of climate change on forest communities. Acknowledgements The authors would like to thank Dr. Paul Sanborn, Dr. Art Fredeen, Dr. Mike Rutherford, and Craig DeLong (British Columbia Ministry of Forests) for discussion and practical input into this project. We also thank Jennifer Catherall for field and lab assistance, Kei Fujimura, Susan Gibson, and Linda Rehaume for lab assistance, and Cameron Grose for assisting with computer issues. Funding for this research was provided by a Natural Sciences and Engineering Research Council of Canada (NSERC) grant to H.B.M. Three anonymous reviewers provided helpful comments on the manuscript. References Agerer, R. (Editor) Colour atlas of ectomycorrhizae. Einhorn-Verlag Eduard Dietenberger, Schwäbisch Gmünd, Germany. Allen, M.F., Swenson, W., Querejeta, J.I., Egerton-Warburton, L.M., and Treseder, K.K Ecology of mycorrhizae: a conceptual framework for complex interactions among plants and fungi. Annu. Rev. Phytopathol. 41: Arocena, J.M., Glowa, K.R., Massicotte, H.B., and Lavkulich, L Chemical and mineral composition of ectomycorrhizosphere soils of subalpine fir (Abies lasiocarpa (Hook.) Nutt.) in the Ae horizon of a luvisol. Can. J. Soil Sci. 79: Baldwin, Q.F Effects of prescribed burning upon mycorrhizal fungal diversity inhabiting the roots of two and a half-year-old black spruce (Picea mariana): molecular characterization of ectomycorrhizal fungi via PCR/ RFLP. M.Sc. thesis, Memorial University of Newfoundland, St. John s, Nfld. Brundrett, M Mycorrhizas in natural ecosystems. Adv. Ecol. Res. 21: Bruns, T.D Thoughts on the processes that maintain local species diversity of ectomycorrhizal fungi. Plant Soil, 170: Burton, P.J., Messier, C., Weetman, G.F., Prepas, E.E., Adamowicz, W.L., and Tittler, R The current state of boreal forestry and the drive for change. In Towards sustainable management of

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